Magnetic particles are known for use in laboratory and industrial procedures in which such particles are stationed or transported by applied magnetic fields. Where each particle is coupled to a selected biologically-active component, either as a matrix material or as a coating for the magnetizable particles, such particles may be effectively used in separatory procedures such as immunoassays and cell separations.
For example, solid-phase radioimmunoassay systems have been reported based on the use of antibodies covalently linked to polymer-coated iron oxide particles. An electromagnet is employed both to mix the particles during incubation (by switching the field on and off) and to separate the antibody-bound and free fractions. Nye, L., G. C. Forrest, H. Greenwood, J. S. Gardner, R. Jay, J. R. Roberts, and J. Landon, Solid-Phase, Magnetic Particle Radioimmunoassay, Clinica Chimica Acta, 69:387-396 (1976). Such techniques have not been free of problems and complications, however. Because of their relatively large size (well in excess of one micron) and high density (of the order of 5 gm/cc), the particles tend to settle rapidly under gravity unless vigorously stirred and also to "crush" large organic molecules. To reduce such problems, it has been proposed that such particles be formed as composites with the magnetizable component supporting, or supported by, a relatively low density non-magnetic component. See U.S. Pat. Nos. 4,177,253, 4,141,687, 4,115,534. By selecting a nonmagnetizable polymeric component having a density that will result in a composite particle whose density approximates that of the aqueous medium to be used, the stability of the suspension may be enhanced.
While the effective density of composite particles may thus be adjusted to suit the suspending medium, the relatively large size of such particles remains a disadvantage because of the smaller surface area per volume as compared with particles in the sub-micron size range. Also, in accordance with Stokes' Law on sedimentation, the sedimentation rate decreases with size; hence, particles of a size of 0.1 micron or less might (in the absence of aggregation) remain in suspension for periods of sufficient duration without continuous agitation, even though the density of the magnetizable material is substantially greater than that of the suspending medium. Unfortunately, small particles continually aggregate in a suspending medium in accordance with von Smoluchovski's Law of flocculation. That law neglects any gravitational or magnetic forces on the particles. Both would tend to increase the packing density of the particles with time, and the latter would also tend to orient and attract magnetized particles; both effects would accelerate aggregation. The practical result is that it becomes extremely difficult to resuspend sub-micron-sized particles of magnetically-susceptible material, especially particles of 0.1 micron or less in size, after such particles have been magnetically aggregated.
While it is known that certain larger particles in the size range of 1.0 to 4.0 microns may be separated and redistributed following magnetization if each particle is encapsulated in a shell of albumin having a thickness of about 0.1 micron, and that certain ferrofluids (particle sizes of about 0.01 micron) can be kept in suspension much longer than given by von Smoluchovski's Law if the material is chemically altered over the outer 10 to 15 .ANG., applicant is unaware of prior art teaching that magnetizable particles in the sub-micron size range, particularly those smaller than 0.1 micron but of domain size or larger, may be treated so that they may be readily resuspended following magnetic aggregation. Compare: Nakamura, T., K. Konno, and T. Morone, Magneto-Medicine: Biological Aspects of Ferromagnetic Fine Particles, J. App. Physics, 42:1320-1324 (1971); U.S. Pat. No. 4,247,406.
This invention is therefore concerned with coated magnetizable microparticles of an average size no greater than 1.0 micron in diameter, and preferably no greater than 0.1 micron in diameter, that may be magnetically aggregated in aqueous suspension and may then be resuspended without difficulty. Thus, in a biological separation procedure, the coated discrete microparticles may be dispersed in a fluid medium containing a component capable of reacting with the coating material, the particles then be exposed to a magnetic field under conditions causing magnetic aggregation of the particles, the aggregated particles may then be separated from the first liquid medium, introduced into a second liquid medium and resuspended in the absence of a magnetic field.
A major aspect of this invention lies in forming each sub-micron-sized particle so that it has a core composed of a magnetically-responsive material with a Curie temperature within the range of about 5.degree. to 65.degree. C., preferably 30.degree. to 40.degree. C., and a magnetic moment at saturation of at least 2.0.mu..sub.B at 4.2.degree. K. The aggregating and resuspending steps may therefore be undertaken in relation to the Curie temperature. Specifically, magnetic attraction between the particles, even if the cores thereof are formed of a material having ferromagnetic properties, will be dissipated by undertaking the resuspension step at or above the Curie temperature and in the absence of a magnetic field. Conversely, magnetic aggregation of the sub-micron-sized particles may be selectively achieved by exposing the particles to a magnetic field while such particles are held at a temperature approximating, or slightly below, the Curie temperature of the magnetizable core material.
A further aspect of the invention relates to the provision of a non-magnetic coating about each magnetizable core. The coating should preferably have a thickness of at least 100 .ANG. or about 10% of the diameter of each microparticle, and in any event a minimum thickness of at least 10 .ANG., and most effectively comprises a water-insoluble cross-linked polymeric material having chemically reactive groups at the surface thereof. In the best mode known for practicing the invention, particle aggregation is inhibited by charged groups provided by the coating material at the periphery of the particles. The coating may be one or more layers with the exposed surface having whatever biological affinity or reactivity is required for a selected treatment or separation procedure, all as well known in connection with immunoassays, cell separation, and the like.
The core material should be ferromagnetic or ferrimagnetic at temperatures below Curie temperature, relatively brittle and not too hard (its hardness should not exceed 7, and preferably 6, on the Mohs scale), and generally compatible with both the coating and the fluid medium in which such particles are to be suspended. As stated, its Curie temperature must be within the range of 5.degree. to 65.degree. C., preferably 30.degree. to 40.degree. C., and the magnetic moment at saturation should be at least 2.0.mu..sub.B at 4.2.degree. K. A lanthanum-manganese-germanium alloy (LaMn.sub.2 Ge.sub.2) has been found effective, but other alloys and oxides having ferromagnetic or ferrimagnetic properties, and Curie temperatures within the specified range, may be used. More specifically, ferrites, yttrium iron garnets, and alloys of RMn.sub.2 X.sub.2, where R is a rare earth and X is germanium or silicon, may be used as long as the specified Curie temperatures and other requirements are met.
Other objects, advantages, and features of the invention will be apparent from the detailed specification.